Janet L. Huebner

Extreme obesity due to impaired leptin signaling in mice does not cause knee osteoarthritis

OBJECTIVE To test the hypothesis that obesity resulting from deletion of the leptin gene or the leptin receptor gene results in increased knee osteoarthritis (OA), systemic inflammation, and altered subchondral bone morphology. METHODS Leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) female mice compared with wild-type mice were studied, to document knee OA via histopathology. The levels of serum proinflammatory and antiinflammatory cytokines were measured using a multiplex bead immunoassay. Cortical and trabecular subchondral bone changes were documented by microfocal computed tomography, and body composition was quantified by dual x-ray absorptiometry. RESULTS Adiposity was increased by approximately 10-fold in ob/ob and db/db mice compared with controls, but it was not associated with an increased incidence of knee OA. Serum cytokine levels were unchanged in ob/ob and db/db mice relative to controls, except for the level of cytokine-induced neutrophil chemoattractant (keratinocyte chemoattractant; murine analog of interleukin-8), which was elevated. Leptin impairment was associated with reduced subchondral bone thickness and increased relative trabecular bone volume in the tibial epiphysis. CONCLUSION Extreme obesity due to impaired leptin signaling induced alterations in subchondral bone morphology without increasing the incidence of knee OA. Systemic inflammatory cytokine levels remained largely unchanged in ob/ob and db/db mice. These findings suggest that body fat, in and of itself, may not be a risk factor for joint degeneration, because adiposity in the absence of leptin signaling is insufficient to induce systemic inflammation and knee OA in female C57BL/6J mice. These results imply a pleiotropic role of leptin in the development of OA by regulating both the skeletal and immune systems.

Diet-induced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice

IntroductionObesity is a major risk factor for the development of osteoarthritis in both weight-bearing and nonweight-bearing joints. The mechanisms by which obesity influences the structural or symptomatic features of osteoarthritis are not well understood, but may include systemic inflammation associated with increased adiposity. In this study, we examined biomechanical, neurobehavioral, inflammatory, and osteoarthritic changes in C57BL/6J mice fed a high-fat diet.MethodsFemale C57BL/6J mice were fed either a 10% kcal fat or a 45% kcal fat diet from 9 to 54 weeks of age. Longitudinal changes in musculoskeletal function and inflammation were compared with endpoint neurobehavioral and osteoarthritic disease states. Bivariate and multivariate analyses were conducted to determine independent associations with diet, percentage body fat, and knee osteoarthritis severity. We also examined healthy porcine cartilage explants treated with physiologic doses of leptin, alone or in combination with IL-1α and palmitic and oleic fatty acids, to determine the effects of leptin on cartilage extracellular matrix homeostasis.ResultsHigh susceptibility to dietary obesity was associated with increased osteoarthritic changes in the knee and impaired musculoskeletal force generation and motor function compared with controls. A high-fat diet also induced symptomatic characteristics of osteoarthritis, including hyperalgesia and anxiety-like behaviors. Controlling for the effects of diet and percentage body fat with a multivariate model revealed a significant association between knee osteoarthritis severity and serum levels of leptin, adiponectin, and IL-1α. Physiologic doses of leptin, in the presence or absence of IL-1α and fatty acids, did not substantially alter extracellular matrix homeostasis in healthy cartilage explants.ConclusionsThese results indicate that diet-induced obesity increases the risk of symptomatic features of osteoarthritis through changes in musculoskeletal function and pain-related behaviors. Furthermore, the independent association of systemic adipokine levels with knee osteoarthritis severity supports a role for adipose-associated inflammation in the molecular pathogenesis of obesity-induced osteoarthritis. Physiologic levels of leptin do not alter extracellular matrix homeostasis in healthy cartilage, suggesting that leptin may be a secondary mediator of osteoarthritis pathogenesis.

Induction of osteoarthritis and metabolic inflammation by a very high‐fat diet in mice: Effects of short‐term exercise

OBJECTIVE To test the hypotheses that obesity due to a very high-fat diet induces knee osteoarthritis (OA), and that short-term wheel-running exercise protects against obesity-induced knee OA by reducing systemic inflammation and metabolic dysregulation. METHODS Male C57BL/6J mice were fed either a control diet (13.5% kcal from fat) or a very high-fat diet (60% kcal from fat) from age 12 weeks to age 24 weeks. From 20 to 24 weeks of age, half of the mice were housed with running wheels. The severity of knee OA was determined by assessing histopathologic features, and serum cytokines were measured using a multiplex bead immunoassay and enzyme-linked immunosorbent assays. Body composition was quantified by dual-energy x-ray absorptiometry, and insulin resistance was assessed by glucose tolerance testing. RESULTS Feeding mice with a very high-fat diet increased knee OA scores and levels of serum leptin, adiponectin, KC (mouse analog of interleukin-8 [IL-8]), monokine induced by interferon-γ (CXCL9), and IL-1 receptor antagonist to an extent in proportion to the gain in body fat (3-fold increase in percent body fat compared to controls). Wheel-running exercise reduced progression of OA in the medial femur of obese mice. In addition, exercise disrupted the clustering of cytokine expression and improved glucose tolerance, without reducing body fat or cytokine levels. CONCLUSION Obesity induced by a very high-fat diet in mice causes OA and systemic inflammation in proportion to body fat. Increased joint loading is not sufficient to explain the increased incidence of knee OA with obesity, as wheel running is protective rather than damaging. Exercise improves glucose tolerance and disrupts the coexpression of proinflammatory cytokines, suggesting that increased aerobic exercise may act independently of weight loss in promoting joint health.

Collagenase 1 and collagenase 3 expression in a guinea pig model of osteoarthritis.

OBJECTIVE To analyze the in vivo compartmental expression of collagenases 1 and 3 (MMP-1 and MMP-13) in the Hartley guinea pig model of spontaneously occurring osteoarthritis (OA) for the purpose of elucidating their roles in the pathogenesis of OA. METHODS Competitive reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry quantification of messenger RNA (mRNA) and protein levels in medial and lateral tibial cartilage obtained from the knee joints of 2-month-old (no OA) and 12-month-old (OA) guinea pigs. RESULTS The patterns of mRNA expression of collagenases 1 and 3 varied with the age of the animal and the compartment of the knee. We also found focal areas of collagenase 1 and collagenase 3 proteins localized to the extracellular matrix of OA lesion sites, coincident with three-quarter/one-quarter collagen cleavage. Collagenase 3 protein was also abundant throughout the medial tibial cartilage of 2-month-old animals. CONCLUSION This represents the first description of bona fide collagenase 1 in a rodent species. Recent evidence, however, based on analysis of mitochondrial DNA homologies, suggests that the guinea pig is not a member of the order Rodentia and may be more closely allied with lagomorphs. This taxonomic controversy leaves open to question the issue of the expression of collagenase 1 in other rodents, such as mice and rats. The presence of active collagenases 1 and 3 at OA lesion sites is consistent with an important role of these enzymes in the cartilage degradation of OA in guinea pigs. The expression of collagenase 3 in medial tibial cartilage from 2-month-old guinea pigs may signify a role of this enzyme in cartilage remodeling with growth and development, or it may represent an early molecular manifestation of OA.

Pain coping skills training and lifestyle behavioral weight management in patients with knee osteoarthritis: A randomized controlled study

Summary Combined training in pain and weight management in overweight and obese OA patients resulted in improved pain and other outcomes compared to either training alone. ABSTRACT Overweight and obese patients with osteoarthritis (OA) experience more OA pain and disability than patients who are not overweight. This study examined the long‐term efficacy of a combined pain coping skills training (PCST) and lifestyle behavioral weight management (BWM) intervention in overweight and obese OA patients. Patients (n = 232) were randomized to a 6‐month program of: 1) PCST + BWM; 2) PCST‐only; 3) BWM‐only; or 4) standard care control. Assessments of pain, physical disability (Arthritis Impact Measurement Scales [AIMS] physical disability, stiffness, activity, and gait), psychological disability (AIMS psychological disability, pain catastrophizing, arthritis self‐efficacy, weight self‐efficacy), and body weight were collected at 4 time points (pretreatment, posttreatment, and 6 months and 12 months after the completion of treatment). Patients randomized to PCST + BWM demonstrated significantly better treatment outcomes (average of all 3 posttreatment values) in terms of pain, physical disability, stiffness, activity, weight self‐efficacy, and weight when compared to the other 3 conditions (Ps < 0.05). PCST + BWM also did significantly better than at least one of the other conditions (ie, PCST‐only, BWM‐only, or standard care) in terms of psychological disability, pain catastrophizing, and arthritis self‐efficacy. Interventions teaching overweight and obese OA patients pain coping skills and weight management simultaneously may provide the more comprehensive long‐term benefits.

Intra-articular delivery of purified mesenchymal stem cells from C57BL/6 or MRL/MpJ superhealer mice prevents posttraumatic arthritis.

Joint injury dramatically enhances the onset of osteoarthritis (OA) and is responsible for an estimated 12% of OA. Posttraumatic arthritis (PTA) is especially common after intra-articular fracture, and no disease-modifying therapies are currently available. We hypothesized that the delivery of mesenchymal stem cells (MSCs) would prevent PTA by altering the balance of inflammation and regeneration after fracture of the mouse knee. Additionally, we examined the hypothesis that MSCs from the MRL/MpJ (MRL) “superhealer” mouse strain would show increased multilineage and therapeutic potentials as compared to those from C57BL/6 (B6) mice, as MRL mice have shown exceptional in vivo regenerative abilities. A highly purified population of MSCs was prospectively isolated from bone marrow using cell surface markers (CD45-/TER119-/PDGFRa+/Sca-1+). B6 MSCs expanded greater than 100,000-fold in 3 weeks when cultured at 2% oxygen and displayed greater adipogenic, osteogenic, and chondrogenic differentiation as compared to MRL MSCs. Mice receiving only a control saline injection after fracture demonstrated PTA after 8 weeks, but the delivery of 10,000 B6 or MRL MSCs to the joint prevented the development of PTA. Cytokine levels in serum and synovial fluid were affected by treatment with stem cells, including elevated systemic interleukin-10 at several time points. The delivery of MSCs did not reduce the degree of synovial inflammation but did show increased bone volume during repair. This study provides evidence that intra-articular stem cell therapy can prevent the development of PTA after fracture and has implications for possible clinical interventions after joint injury before evidence of significant OA.

Absence of posttraumatic arthritis following intraarticular fracture in the MRL/MpJ mouse

OBJECTIVE Posttraumatic arthritis is a frequent long-term complication of intraarticular fractures. A model of a closed intraarticular fracture in C57BL/6 mice that progresses to posttraumatic arthritis has been developed. The MRL/MpJ mouse has shown unique regenerative abilities in response to injury. The objective of this study was to determine if the MRL/MpJ mouse is protected from posttraumatic arthritis after intraarticular fractures. METHODS Intraarticular fractures were created in MRL/MpJ mice and C57BL/6 control mice (n = 16 each). Limbs were analyzed for posttraumatic arthritis 4 and 8 weeks after fracture using microfocal computed tomography bone morphology, subchondral bone thickness evaluation, and histologic evaluation of cartilage degeneration. Serum cytokines and biomarkers were measured after the mice were killed. RESULTS Intraarticular fractures were successfully created in all 32 mice. In the experimental fractured limbs, C57BL/6 mice had a decrease in bone density, increased subchondral bone thickness, and increased cartilage degeneration compared with normal contralateral control limbs. In the MRL/MpJ mice, no differences in bone density, subchondral bone thickness, or histologic grading of cartilage degeneration were seen between fractured and contralateral control limbs. Cytokine analysis showed lower systemic levels of the proinflammatory cytokine interleukin-1alpha (IL-1alpha) and higher levels of the antiinflammatory cytokines IL-4 and IL-10 in the MRL/MpJ mice. CONCLUSION This study shows that the MRL/MpJ mouse is relatively protected from posttraumatic arthritis after intraarticular fracture. Further investigation into the mechanism involved in this response will hopefully provide new insight into the pathogenesis, prevention, and treatment of posttraumatic arthritis after intraarticular fracture.

Acute joint pathology and synovial inflammation is associated with increased intra-articular fracture severity in the mouse knee

OBJECTIVE Post-traumatic arthritis is a frequent cause of disability and occurs most commonly and predictably after articular fracture. The objective of this investigation was to examine the effect of fracture severity on acute joint pathology in a novel murine model of intra-articular fracture. DESIGN Low and high energy articular fractures (n=25 per group) of the tibial plateau were created in adult male C57BL/6 mice. The acute effect of articular fracture severity on synovial inflammation, bone morphology, liberated fracture area, cartilage pathology, chondrocyte viability, and systemic cytokines and biomarkers levels was assessed at 0, 1, 3, 5, and 7 days post-fracture. RESULTS Increasing intra-articular fracture severity was associated with greater acute pathology in the synovium and bone compared to control limbs, including increased global synovitis and reduced periarticular bone density and thickness. Applied fracture energy was significantly correlated with degree of liberated cortical bone surface area, indicating greater comminution. Serum concentrations of hyaluronic acid (HA) were significantly increased 1 day post-fracture. While articular fracture significantly reduced chondrocyte viability, there was no relationship between fracture severity and chondrocyte viability, cartilage degeneration, or systemic levels of cytokines and biomarkers. CONCLUSIONS This study demonstrates that articular fracture is associated with a loss of chondrocyte viability and increased levels of systemic biomarkers, and that increased intra-articular fracture severity is associated with increased acute joint pathology in a variety of joint tissues, including synovial inflammation, cortical comminution, and bone morphology. Further characterization of the early events following articular fracture could aid in the treatment of post-traumatic arthritis.

Diet‐induced obesity significantly increases the severity of posttraumatic arthritis in mice

OBJECTIVE Obesity and joint injury are primary risk factors for osteoarthritis (OA) that involve potential alterations in the biomechanical and inflammatory environments of the joint. Posttraumatic arthritis is a frequent long-term complication of intraarticular fractures. Obesity has been linked to primary OA and may potentially contribute to the development of posttraumatic arthritis by a variety of mechanisms. The objectives of this study were to determine whether diet-induced obesity influences the severity of posttraumatic arthritis in mice and to examine the interrelationships between joint degeneration and serum levels of the inflammatory cytokines and adipokines that are involved in this response. METHODS C57BL/6 mice were fed either normal chow (13% fat) or a high-fat diet (60% fat) starting at 4 weeks of age. At 16 weeks of age, half of the mice in each group were subjected to a closed intraarticular fracture of the left knee. At 8 weeks postfracture, knee OA was assessed by cartilage and synovium histology in addition to bone morphology. Serum cytokine concentrations were determined with multiplex assays. RESULTS Fractured knee joints of mice receiving a high-fat diet showed significantly increased OA degeneration compared with nonfractured contralateral control knees, while fractured knee joints of mice receiving a low-fat diet did not demonstrate significant differences from nonfractured contralateral control knees. A high-fat diet increased serum concentrations of interleukin-12p70 (IL-12p70), IL-6, and keratinocyte-derived chemokine while decreasing adiponectin concentrations. Joint injury also increased IL-12p70 concentrations in mice receiving a high-fat diet. Systemic levels of adiponectin were inversely correlated with synovial inflammation in control limbs. CONCLUSION Diet-induced obesity significantly increased the severity of OA following intraarticular fracture. Obesity and joint injury together can alter systemic levels of inflammatory cytokines such as IL-12p70.

Dietary fatty acid content regulates wound repair and the pathogenesis of osteoarthritis following joint injury

Objective The mechanisms linking obesity and osteoarthritis (OA) are not fully understood and have been generally attributed to increased weight, rather than metabolic or inflammatory factors. Here, we examined the influence of fatty acids, adipokines, and body weight on OA following joint injury in an obese mouse model. Methods Mice were fed high-fat diets rich in various fatty acids (FA) including saturated FAs (SFAs), ω-6 polyunsaturated FAs (PUFAs), and ω-3 PUFAs. OA was induced by destabilising the medial meniscus. Wound healing was evaluated using an ear punch. OA, synovitis and wound healing were determined histologically, while bone changes were measured using microCT. Activity levels and serum cytokines were measured at various time-points. Multivariate models were performed to elucidate the associations of dietary, metabolic and mechanical factors with OA and wound healing. Results Using weight-matched mice and multivariate models, we found that OA was significantly associated with dietary fatty acid content and serum adipokine levels, but not with body weight. Furthermore, spontaneous activity of the mice was independent of OA development. Small amounts of ω-3 PUFAs (8% by kcal) in a high-fat diet were sufficient to mitigate injury-induced OA, decreasing leptin and resistin levels. ω-3 PUFAs significantly enhanced wound repair, SFAs or ω-6 PUFAs independently increased OA severity, heterotopic ossification and scar tissue formation. Conclusions Our results indicate that with obesity, dietary FA content regulates wound healing and OA severity following joint injury, independent of body weight, supporting the need for further studies of dietary FA supplements as a potential therapeutic approach for OA.